The DELFIA time-resolved fluorescence assay for inhibition of poly(ADP-ribose) polymerase (PARP) is a 96-well microplate assay that measures the inhibition of PARP-activated incorporation of biotinylated nicotinamide adenine diphsophate (NAD). The incorporation is detected using Europiumlabelled streptavidin. The method is a convenient way to screen test compounds for PARP activity in high throughput primary screening and secondary screening.
Poly(ADP-ribose) polymerase (PARP) is a nuclear protein involved in the response to DNA damage, where it catalyzes the polymerization of nicotinamide adenine diphsophate (NAD) into chains of poly(ADPribose) polymers1. The polymerization occurs on a number of nuclear proteins, as well as on the PARP protein itself. Following activation by DNA strand breaks, PARP hydrolyzes NAD and catalyzes the formation of PARP onto itself and other nuclear proteins, with the release of nicotinamide. PARP binds to singleand double-stranded DNA breaks. When the enzyme undergoes auto-poly (ADPribosyl) ation, the automodified PARP dissociates from the DNA and becomes inactive2.
Inhibitors of PARP have been shown to help prevent ischemic injury in the brain, increase apoptosis in cancer cells, prevent infiltration of neutrofils and subsequent inflammation, and to suppress the production of nitric oxide synthase in macrophages3,4. In response to low to moderate levels of DNA damage, PARP is activated and NAD levels in the cells decrease. Experiments with PARP inhibitors suggest that PARP activity may be necessary to rescue cells after low to moderate levels of DNA damage. In contrast, when cells experience massive levels of DNA damage and DNA strand breaks, activation of PARP can lead to depletion of NAD and ATP, resulting in a marked decrease in energydependent processes and in DNA repair. In this situation, activation of PARP by massive DNA damage processes